干扰山羊KLF12促进皮下脂肪细胞分化

doi:10.3864/j.issn.0578-1752.2022.01.015

摘要/Abstract

摘要：

【背景】脂肪组织分为皮肤下的皮下脂肪组织（subcutaneous adipose tissue,SAT）和腹部内器官周围的内脏脂肪组织（visceral adipose tissue,VAT）,皮下脂肪作为影响肉类美味与否的重要因素,探究皮下脂肪沉积分子调控机制对于育种改良和畜牧业的发展至关重要。Krüppel-like factors 12 (KLF12)是一个进化保守的转录因子,可以在多种细胞类型中表达并控制着广泛的细胞过程。【目的】研究获得山羊KLF12的分子特征并进行生物信息学分析,同时明确KLF12在山羊组织和细胞中的表达模式以及干扰KLF12对山羊皮下脂肪细胞分化的调控作用,为进一步研究KLF12在脂肪沉积过程中的潜在作用提供理论依据。【方法】利用逆转录PCR（reverse transcription PCR,RT-PCR）方法克隆山羊KLF12完整编码序列（coding sequence,CDS）区,使用在线生物信息学分析软件对山羊KLF12核苷酸序列和氨基酸序列进行分析。利用实时荧光定量PCR（Quantitative Real-time PCR,qRT-PCR）技术检测KLF12在山羊心脏、肝脏、腹部脂肪、皮下脂肪、臂三头肌、背最长肌等14个组织中的表达水平,以及诱导分化不同时间段KLF12在皮下前体脂肪细胞中的表达水平。随后,试验通过化学合成山羊KLF12小干扰RNA（si-KLF12）,使用Lipofectamine RNAiMAX转染试剂将山羊si-KLF12序列转染到体外培养的山羊皮下前体脂肪细胞中。使用100 µmol·L^-1油酸导液诱导脂肪细胞分化。利用油红O以及Bodipy染色方法和qRT-PCR技术分别从形态学以及分子生物学的角度阐明干扰KLF12对皮下前体脂肪细胞脂滴积聚和脂肪分化标志基因mRNA表达水平的影响。【结果】试验成功获得包含开放阅读框（open reading frame,ORF）（1 209 bp）的山羊KLF12（1 315 bp,编码402个氨基酸）。亚细胞定位结果显示KLF12主要位于细胞核,此外,KLF12无跨膜结构域和信号肽,且在317—341、347—371及377—399氨基酸处存在3个典型的锌指结构域（ZnF_C2H2）。组织表达谱结果显示KLF12在山羊心脏和脾脏的表达水平极显著高于其他组织（P<0.01）。此外,在山羊皮下前体脂肪细胞分化过程中KLF12表达水平在诱导分化60 h时达到峰值。于山羊皮下前体脂肪细胞中转染si-KLF12后利用油红O以及Bodipy染色法从形态学观察发现脂肪细胞脂滴聚积明显增加,同时qRT-PCR结果显示,脂肪分化标志基因脂蛋白脂肪酶（lipoprteinlipase,LPL）和过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor γ,PPARγ）的表达水平显著升高（P<0.05）而前脂肪细胞生长因子（preadipocyte factor 1,Pref-1）的表达水平极显著降低（P<0.01）。结合形态学观察结果以及脂肪分化标志基因表达水平变化情况,推测KLF12在皮下脂肪细胞分化过程中起到负调控作用。【结论】通过对山羊KLF12的分子生物学特征、组织细胞间的表达规律以及对山羊皮下脂肪细胞分化过程的潜在调控作用的研究表明,KLF12是山羊皮下脂肪细胞分化过程中的负调控因子,并且这种作用可能是通过调控LPL、PPARγ和Pref-1实现的,为进一步探究KLF12在调控脂肪细胞分化过程中的分子机制奠定了基础。

关键词: 山羊, KLF12, 分子特征, 干扰, 皮下脂肪细胞

Abstract:

【Background】Subcutaneous adipose tissue (SAT) under the skin is an important factor affecting the taste of meat. Exploring the molecular regulation mechanisms of SAT deposition is very important for breeding improvement and the development of animal husbandry. Krüppel-like factors 12 (KLF12) is a conserved transcription factor that evolutionarily conserved, and it was found that it could be expressed in a variety of cell types and control a wide range of cellular processes. 【Objective】 This study aimed to obtain the coding sequence (CDS) of goat KLF12 and to explore its molecular characteristics. Moreover, the study also intended to clarify the expression pattern of KLF12 in goat tissues and subcutaneous adipocytes, and to explore the role of KLF12 in goat subcutaneous preadipocytes differentiation via interference KLF12, so as to provide a theoretical basis for further research on the potential role of KLF12 in the process of fat deposition. 【Method】 In this study, the goat KLF12 CDS sequence was cloned by Reverse Transcription PCR ( RT-PCR) method, and the nucleotide sequence and amino acid sequence of goat KLF12 were analyzed on online bioinformatics analysis software. The Quantitative Real-time PCR (qRT-PCR) technology was used to detect the expression levels of KLF12 in goat heart, liver, abdominal fat, subcutaneous fat, triceps brachii, longissimus dorsi and other 14 tissues. Furthermore, the expression level of KLF12 in subcutaneous preadipocytes in different differentiation periods was investigated. Then, the goat KLF12 small interfering RNA (si-KLF12) was chemically synthesized and transfected into goat subcutaneous preadipocyte in vitro by using Lipofectamine RNAiMAX transfection reagent. Subsequently, 100 µmol·L^-1 oleic acid induced adipocyte differentiation. Oil red O and Bodipy staining methods and qRT-PCR techniques were used to clarify the effects of interference KLF12 on the accumulation of lipid droplets in subcutaneous preadipocytes and the mRNA expression levels of adipose differentiation marker genes from the perspectives of morphology and molecular biology. 【Result】 The goat KLF12 (1 315 bp) were successfully obtained, which contained an Open Reading Frame (ORF) (1 209 bp) and encoded 402 amino acids. The subcellular localization results showed that KLF12 was mainly located in the nucleus. In addition, KLF12 had no transmembrane domain and signal peptide but 3 typical zinc finger domains (ZnF_C2H2) at amino acids 317-341, 347-371 and 377-399. Tissue expression profile showed that the expression level of KLF12 in goats’ heart and spleen were significantly higher than that in other tissues (P<0.01). Moreover, during subcutaneous preadipocytes differentiation, the expression level of KLF12 was peaked at 60 h. After transfection of si-KLF12 into goat subcutaneous preadipocytes, the results of oil red O and Bodipy saining showed that accumulation of lipid droplets in adipocytes were significantly increased. At the same time, the results of qRT-PCR showed that the expression levels of key adipogenic regulatory genes like lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor γ (PPARγ) were significantly increased (P<0.05), while the expression level of preadipocyte growth factor (Pref-1) was extremely significantly reduced (P<0.01). Combined with the morphological observation results and the changes in the expression levels of key adipogenic regulatory genes, it was speculated that KLF12 played a negative regulatory role in the differentiation of subcutaneous adipocytes. 【Conclusion】 By investigating the basic molecular biological characteristics and its expression pattern between tissues and cells of goat KLF12 and analyzing the potential regulatory effects of KLF12 on differentiation process of goat subcutaneous adipocytes, the results suggested that KLF12 played a negative role in goats subcutaneous preadipocytes differentiation, and this effect achieved by regulating LPL, PPARγ and Pref-1, which laid a foundation for further exploring the molecular mechanism of KLF12 in regulating the differentiation of adipocytes.

Key words: goat, KLF12, molecular characteristics, knockdown, subcutaneous adipocytes

杜宇,王永,孟庆勇,朱江江,林亚秋. 干扰山羊KLF12促进皮下脂肪细胞分化[J]. 中国农业科学, 2022, 55(1): 184-196.

DU Yu,WANG Yong,MENG QingYong,ZHU JiangJiang,LIN YaQiu. Knockdown Goat KLF12 to Promote Subcutaneous Adipocytes Differentiation[J]. Scientia Agricultura Sinica, 2022, 55(1): 184-196.

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名称 Name	引物序列5°至3° Primer Sequence	退火温度 Tm (℃)	产物长度 Products length (bp)	用途 Purpose	登录号 miRbase ID
KLF12	S: TTAGCGCATCATGTGATCCG A: TGGGGTGCCGCTAAGAGAT	58	1315	RT-PCR	XM_005687692.2
KLF12	S: TCTAAGGTCACATTTGGCAGGTC A: CCAATCGGTGCCTGTTGTCTAC	61	183	qRT-PCR	KX247669.1
UXT	S: GCAAGTGGATTTGGGCTGTAAC A: ATGGAGTCCTTGGTGAGGTTGT	60	180	qRT-PCR	XP_005700899.1

名称 Name	引物序列5°至3° Primer sequence	退火温度 Tm (℃)	产物长度 Products length (bp)	用途 Purpose	登录号 miRbase ID
si-KLF12	UGGACAAGUCCACUGGCUCAGUUUG			siRNA
Negative Control	S: UUCUCCGAACGUGUCACGUTT A: ACGUGACACGUUCGGAGAATT			RNAi
LPL	S: TCCTGGAGTGACGGAATCTGT A: GACAGCCAGTCCACCACGAT	60	174	qPCR	NM_001285607.1
PPARγ	S: AAGCGTCAGGGTTCCACTATG A: GAACCTGATGGCGTTATGAGAC	60	197	qPCR	NM_001285658.1
AP2	S: TGAAGTCACTCCAGATGACAGG A: TGACACATTCCAGCACCAGC	58	143	qPCR	NM_001285623.1
C/EBPβ	S: CAAGAAGACGGTGGACAAGC A: AACAAGTTCCGCAGGGTG	66	204	qPCR	XM_018058020.1
Pref1	S: CCGGCTTCATGGATAAGACCT A: GCCTCGCACTTGTTGAGGAA	65	178	qPCR	KP686197.1
SREBP1	S: AAGTGGTGGGCCTCTCTGA A: GCAGGGGTTTCTCGGACT	58	127	qPCR	NM_001285755.1
UXT	S: GCAAGTGGATTTGGGCTGTAAC A: ATGGAGTCCTTGGTGAGGTTGT	60	180	qPCR	XP_005700899.1